Short-Term Fostamatinib Treatment Impacts Hematopoiesis and Downregulates Thromboinflammation in Townes Sickle Mice

Background:

Vaso-occlusive crises (VOC) coupled with thrombosis, inflammation and immune dysregulation contribute to organ damage and poor outcomes in sickle cell disease (SCD). VOCs are promoted by sickle RBC adhesion to the endothelium driven by elevated neutrophil count and dysregulated neutrophil extracellular trap formation (NETosis). NETosis, triggered by signaling pathways involving FcγR receptors and integrins crucially involves the cytoplasmic protein spleen tyrosine kinase (SYK). We investigated whether therapeutically targeting SYK-mediated neutrophil activation reduces thromboinflammation and VOC in SCD.

Methods:

We treated Townes sickle mice (20±2 weeks-old; genotype SS) with either vehicle (n=11) or fostamatinib (R788) containing chow [two dosages and durations: 3g/kg for 14 days (n=7) and 7 days (n=3); and 2g/kg for 14 days (n=4) and 7 days (n=4)]. We evaluated the safety of fostamatinib treatment by assessing complete blood count, liver function and spleen weight. The SYK-inhibiting effects of Fostamatinib on neutrophils were assessed by flow cytometric evaluation of SYK phosphorylation (p-SYK), and NETosis, in Fostamatinib-treated and vehicle-treated mice. These assessments were conducted at baseline (after treatment), as well as following hypoxia-reperfusion (H/R) and intraperitoneal TNF injection to model VOC and inflammation observed in humans with SCD. Data are presented as mean±SD comparing Fostamatinib vs. vehicle-treated mice (p < 0.05*, p < 0.01**, p < 0.001***, p < 0.0001****).

Results:

Compared to vehicle-treated, Fostamatinib-treated mice exhibited significantly greater anemia across all treatment doses and durations (Hb g/dL: vehicle 8.7±1.1; 3g/kg-14-Day 5.5±1.6***; 2g/kg-14-Day 6.9±1.7*; 3g/kg-7-Day 5.7±2.4**; 2g/kg-7-Day 7.1±0.9*). White blood cell counts were significantly reduced with 14 days of exposure for both doses, but not with 7 days of exposure (k/mcL: vehicle 33.3±10.1; 3g/kg-14-Day 15.3±7.2***; 2g/kg-14-Day 15.0±1.4**; 3g/kg-7-Day 28.6±11.6; 2g/kg-7-Day 30.6±3.3). Neutrophil counts were significantly reduced with all treatments except the 2g/kg-7-Day regimen (k/mcL: placebo 4.0±1.0; 3g/kg-14-Day 1.4±1.0****; 2g/kg-14-Day 1.9±1.0**; 3g/kg-7-Day 1.4±0.1***). Although liver enzymes (AST, ALT) and markers of hemolysis (LDH) were not impacted, total bilirubin levels were consistently elevated across all doses and durations (mg/dL: vehicle 1.4±0.5; 3g/kg-14-Day 2.6±1.2*; 2g/kg-14-Day 3.9±1.5**; 3g/kg-7-Day 4.1±2.5**; 2g/kg-7-Day 3.8±0.9***).

Basal NET formation was similar after both Fostamatinib and vehicle treatment (%NETosis, vehicle 8±5; 3g/kg-14-Day 5±5; 2g/kg-14-Day 7±2; 3g/kg-7-Day 10±2; 2g/kg-7-Day 8±7). Neutrophil SYK phosphorylation was significantly reduced by treatment with 2g/kg Fostamatinib for 7 days compared to vehicle (p-SYK, mean fluorescence intensity (MFI), vehicle (n=7) 1010±152; 2g/kg-7Day (n=6) 767±102*). However, Hypoxia-reoxygenation challenge led to exaggerated NET formation compared to baseline (p<0.0001), which was unaffected by Fostamatinib treatment (% NETosis, vehicle 44±10; 2g/kg-7Day 55±9) despite continuous inhibition of neutrophil SYK phosphorylation post-Hypoxia-reperfusion (p-SYK MFI, vehicle 1018±26; 2g/kg-7D 935±9**). Intraperitoneal TNF-injection was followed by exaggerated NET formation in vehicle-treated mice and was attenuated in Fostamatinib treated mice although the difference was not significant (% NETosis n=3 each, vehicle 41±26; 3g/kg-14Day 5±7; p=0.08).

Massive splenomegaly reflective of extramedullary hematopoiesis in SS mice was significantly reduced across all treatment doses and durations except for the lowest dosage (spleen-to-body weight ratio (%): placebo 7.8±0.7; 3g/kg-14-Day 4.2±1.0****; 2g/kg-14-Day 4.0±1.0****; 3g/kg-7-Day 5.9±1.1**; 2g/kg-7-Day 7.0±1.6). Histopathological examination of the spleen revealed a trend toward increased hemosiderin deposition and fewer splenic infarcts in fostamatinib-treated mice.

Conclusions:

Targeting SYK with fostamatinib appeared to reduce thromboinflammatory responses but did not affect hypoxia-induced neutrophil activation in Townes sickle mice. Severe anemia and decreased leukocytosis suggest suppression of splenic hematopoiesis, indicating that additional research is needed to clarify the effects of this treatment in humans with SCD.

No relevant conflicts of interest to declare.

Fostamatinib (R788; active moiety, R406) is a medication used to treat chronic immune thrombocytopenia (ITP) in adults who haven't responded to other treatments. It works by inhibiting spleen tyrosine kinase (SYK), which helps reduce the destruction of platelets by the immune system. Fostamatinib is taken orally and may cause side effects such as diarrhea, nausea, and liver enzyme abnormalities. Fostamatinib is shown to be effective for treating COVID-19 patients due to its potential to reduce inflammation and neutrophil extracellular trap formation (NETosis) by inhibiting neutrophil SYK. NETosis is a driver that promote sickle RBC adhesion to the endothelium and the occurrence of frequent vaso-occlusive crises (VOC) in sickle cell disease (SCD). Frequent VOCs exacerbate inflammation and immune dysregulation and contribute to organ damage and poor outcomes in SCD. We explored the role of Fostamatinib in controlling dysregulated neutrophil activity in Sickle Cell Disease to control SCD-associated thromboinflammation using Townes sickle mice model.